In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of non-productive and staggered productive intermediates arise at different reprogramming time points1,2,3,4,5,6,7,8,9,10,11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13, prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that, during reprogramming, cells progressively lose donor cell identity and gradually acquire iPS cell properties1,2,7,8,10. Here we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen, we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200, that are absent in both fibroblasts and iPS cells. Single-cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic, reprogramming phase2. Expression profiling reveals early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1 (also known as Zfp42), Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus represent some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition.
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We thank P. Lovelace, R. Finck, K. M. Loh, K. Tanabe and S. Marro for advice. We thank J. Hanna for the secondary Mbd3fl/− MEFs. We also thank S. Knöbel for the mEF-SK4 antibody. This work was supported by the California Institute of Regenerative Medicine grant RB2-01592 (to G.P.N.), the Institute for Stem Cell Biology and Regenerative Medicine at Stanford, and a New York Stem Cell Foundation-Robertson Investigator Award. E.L. was supported by the California Institute for Regenerative Medicine Predoctoral Fellowship TG2-01159 and National Science Foundation Graduate Research Fellowship DGE-114747. E.R.Z. was supported by National Institutes of Health National Research Service Award F32 GM093508-01. M.W. is a New York Stem Cell Foundation-Robertson Investigator and a Tashia and John Morgridge Faculty Scholar at the Child Health Research Institute at Stanford.
The authors declare no competing financial interests.
Extended data figures and tables
a, Shown are surface markers detected in MEFs, partially reprogrammed cells (PR) or ESCs analysed by flow cytometry. Numbers indicate the percentage of each population positive for the marker of interest, relative to isotype control samples. Markers are grouped for enrichment in single populations or shared between multiple populations. b, SPADE analysis for MEFs, mESCs and PRCs for surface markers analysed by mass cytometry (continued from Fig. 1b). Colour bars (bottom) represent ArcSinh-transformed counts for each marker.
a, SPADE analysis of lentiviral-infected MEF reprogramming populations analysed by mass cytometry (continued from Fig. 1c). Colours bars (bottom) represent ArcSinh-transformed counts for each marker. b, Biaxial plots for selected markers in control populations and during MEF reprogramming.
a, SPADE analysis of lentiviral-infected MEF reprogramming populations analysed by mass cytometry (continued from Fig. 1c). Colours bars (bottom) represent ArcSinh-transformed counts for each marker. b, Day 12 and 16 time points for markers shown in Fig. 1c). Coloured bars for percentage total represent absolute percentages.
a, Ninety-six-well reprogramming assay. Twenty cells per well sorted at days 3, 6 and 9. Sox2–eGFP+ colonies were assayed on day 24. b, Gating strategy for SSEA1 in controls and day 6 reprogramming population. High- and low-expressing populations were determined on the basis of MEF and ESC control levels. c, Ten thousand SSEA1high (black bar) or SSEA1low (white bar) were sorted onto 3 cm gelatinized plates with feeders. Sox2–eGFP+ colonies were counted on day 24 (n = 3 independent experiments). d, Gating strategy for CD73 and CD49d in controls and day 6 reprogramming population. High- and low-expressing populations were determined on the basis of MEF, PRC and ESC control levels. e, f, Treatment of reprogramming populations with compounds affecting CD73 and CD49d. Shown are 96-well reprogramming efficiencies for infected Rosa-rtTA Sox2–eGFP MEFs (e) or secondary Mbd3fl/− MEFs (f). The y axis displays wells with Sox2–eGFP+ colonies 24 days after infection (e) or wells with Oct4–eGFP+ colonies 8 days after transgene induction (f) and treated with the indicated compounds for the days (D) indicated (n = 2 independent experiments).
a, Schematic of continuation analysis. Reprogramming populations were sorted for poised (CD73high or CD49high) and non-poised (CD73low) populations on day 6, cultured for 10 days on a 3 cm plate and analysed by mass cytometry on day 16. b, Morphology of CD49dhigh, CD73high or CD73low cells sorted on day 6 and inspected on day 10. Poised CD49dhigh or CD73high cells form compact colonies within several days of sorting while non-poised CD73low cells fail to do so. c, SPADE analysis (day 16) of cells sorted at day 6 for CD73high/CD49dhigh CD73high, CD49high and CD73low expression (continued from Fig. 3h). Boxes highlight a SSEA1high CD326high branch that is unique to the poised populations. Colours bars (bottom) ArcSinh-transformed counts for each marker.
Extended Data Figure 6 Continuation analysis replicates confirm a SSEA1high CD326high branch that is unique to poised populations.
Continuation analysis replicates for reprogramming-prone (CD73high/CD49dhigh, CD73high, CD49dhigh) and non-prone (CD73low) populations. Boxes highlight a SSEA1high CD326high branch that is unique to the poised populations. Colours bars (bottom) represent absolute percentages (left panel) and ArcSinh-transformed counts for each marker.
a, Genes differentially expressed between reprogramming-prone (day 6 or day 9 CD73high or CD49dhigh) and non-prone (CD73low) populations. Genes with more than twofold differential expression between reprogramming-prone and non-prone were selected and k-means clustered (k = 5) with control and total reprogramming population expression values. b, Heat map of pluripotency-associated genes shown in Fig. 4a (log2). c, d, Quantitative PCR verification of Etv5 (c) and Nr0b1 (d) expression levels (n = 3 technical replicates). e, f, Etv5 (e) and Nr0b1 (f) knockdown qPCRs (n = 3 technical replicates). g, Representative FACS plots for day 9 CD73high/CD49dhigh quantification shown in Fig. 4b. h, Demonstration of ESC self-renewal after infection with Etv5 and Nr0b1 hairpins All infected ESCs continue to express Oct4 after passaging except ESCs infected with shEtv5–8 (n = 1).
a, b, Expression analysis for CD49d, CD73 and CD104 for previously reported highly efficient reprogramming systems generated by transient expression of C/EBPα13 (a) or Mbd3 depletion12 (b). c, Oct4-GFP transgene reporter signal and d, SSEA1 and CD326 levels for the Mbd3fl/− secondary reprogramming MEFs for untreated (left) and 9 days after induction (right). e, SPADE analysis for reprogramming Mbd3fl/− secondary MEFs at days 0, 3, 6, 9 and 12 using all surface markers by mass cytometry. Percentage totals of cells and representative markers are shown for each time point. Remaining markers are shown in Extended Data Figs 9 and 10. Colours bars represent absolute percentages (left) and ArcSinh-transformed counts for each marker. f, Verification of Mbd3 loss in passage 3 Rosa26-CreER, Mbd3fl/− secondary MEFs after treatment with 4OH-tamoxifen. Mbd3 levels were compared with passage 3 Rosa-rtTA± MEFs. While there are several unspecific bands, there is clearly one band around the expected size of Mbd3 absent in 4OH-tamoxifen-treated cells (arrow).
a, SPADE analysis for 2° Mbd3fl/− MEF reprogramming populations (continued from Extended Data Fig. 8e). Colour bars (bottom) represent ArcSinh-transformed counts for each marker. b, Biaxial plots for selected markers.
SPADE analysis for 2° Mbd3fl/−reprogramming populations (continued from Extended Data Fig. 8e). Colours bars (bottom) represent ArcSinh-transformed counts for each marker.
This table contains antibody panels, sheet 1 shows the CyTOF staining panel and sheet 2 shows the FACS staining panel. (XLSX 51 kb)
This table contains cell counts for CyTOF experiments. (XLSX 40 kb)
This table contains hairpin and quantitative PCR sequences. (XLSX 34 kb)
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Lujan, E., Zunder, E., Ng, Y. et al. Early reprogramming regulators identified by prospective isolation and mass cytometry. Nature 521, 352–356 (2015). https://doi.org/10.1038/nature14274
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